Molecular Mechanism Of Wheat Molecular Chaperone HSP90.2 Increasing Grain Weight And Powdery Mildew Resistance

Wheat is an important food crop in our country,and its high yield and stable production are the keys to ensure food security.Thousand kernel weight is an important agronomic trait that affects wheat yield,which is the primary goal of wheat’s high-yield breeding.However,wheat yield is under the threat of fungal diseases such as powdery mildew,and research on powdery mildew resistance plays a decisive role in stabilizing wheat yield.Heat shock protein 90(HSP90)is an essential molecular chaperone protein,which is widely involved in the process of plant disease resistance and stress resistance,but the molecular mechanism that regulates wheat kernel weight and powdery mildew resistance remains elusive.In this research,we cloned and studied wheat HSP90.2-7B to explore its working mechanism to regulate grain weight and powdery mildew resistance.In the mutant library mutagenized by tetraploid wheat Kronos,we found a mutant with reduced grain weight,CO2 assimilation kernel enhanced 1(cake1).The transcriptome sequencing and exon capture technology linked the phenotype with a premature termination in the heat shock protein HSP90.2-7B prematurely.Other prematurely terminated mutants and RNAi transgene interference against the endogenous gene lead to significant grain weight reductions.In contrast,HSP90.2-7B overexpressing has increased grain weight,confirming this gene’s positive regulation on grain weight.HSP90.2 mutants have less chlorophyll content and lower photosynthetic efficiency.HSP90.2-7B binds the photosystem II membrane proteins PsbO and CP43 in vivo and in vitro,which occurs around the chloroplast.This distribution pattern is consistent with the results of HSP90.2 binding to TOC159 in vivo and in vitro.Finally,HSP90.2-7B and the cytoskeleton protein actin can bind TOC159 in the outer chloroplast envelope.suggesting that HSP90.2 is specifically involved in the process of target proteins to enter the chloroplast.To detect the role of HSP90.2 in the process of PsbO entering the chloroplast,we first used specific inhibitors to interfere with the function of HSP90 protein and detected a reduction of PsbO in the chloroplast.The accumulation of PsbO in the chloroplast of the HSP90.2 double mutant decreased,but the content of total protein remained unchanged.The RNAi transgenic wheat of HSP90.2 also showed a similar trend,suggesting that HSP90.2 is specifically involved in the anchor and translocation process of PsbO to enter the chloroplast.Field experiments showed that the psbo-A1 mutant had significant reductions in the grain size,the thousand kernel weight,and plot yield.Based on the above results,we hypothesize that HSP90.2-7B may play a role in transporting nascent peptides from the nuclear-encoded photosystem members to the chloroplast along the cytoskeleton actin to reach TOC159 at the outer membrane transporter complex to facilitate chloroplast-transportation and photosynthesis for the accumulation of grain biomass.HSP90.2-7B has two haplotypes in the resequencing data of the wheat genomes of modern Chinses cultivars.Haplotype II only existed in varieties in the middle and lower part of the Yangtze River,where the pressure of fungal diseases is very high.This suggests that HSP90.2-7B may participate in the wheat disease resistance process.Since most disease-resistant proteins are nuclear-encoded proteins,they are then transported into different organelles(such as chloroplasts)to exert their diseaseresistant functions.We screened a yeast double-hybrid library for the potential clients of HSP90.2.and detected large numbers of target proteins with both the full-length and C-terminal protein of wheat HSP90.2.However,the number of binding proteins in the middle domain is about an order of magnitude lower,indicating the difference of wheat HSP90.2 from animal homologous protein.We analyzed the expression of target protein during pathogen infection but only found a few genes induced by pathogens,suggesting that HSP90.2 assists the maturation of some target proteins to carry out their roles in disease resistance.Among the disease-related clients of HSP90.2,the Pm3 family-like proteins are potential powdery mildew resistance genes,and the 2Q2 member worked as an example for follow-up experimental research.The C-terminal domain of HSP90.2 can bind to the CC-NB-ARC domain of 2Q2 in vivo.The 2Q2-FL and 2Q2-CC-NB-ARC domain mainly existed in the chloroplast.Interference against the normal function of HSP90.2by specific inhibitors or HSP90.2 double mutants effectively inhibits the accumulation of powdery mildew protein 2Q2 in chloroplasts.The 2Q2△LRR transgenic line was susceptible to powdery mildew,suggesting the contribution of 2Q2 to Pm-resistance.The HSP90.2 double mutant has lower Pm-resistance,while HSP90.2-7B overexpression lines have higher.Therefore,the protein binding group of HSP90.2provides alternative strategy for screening disease resistance and stress resistance candidate protein at the translation maturity level,which can supplement differentially expressed genes.In summary,we have conducted genetic and proteomic studies on the wheat molecular chaperone HSP90.2-7B,which provides a theoretical basis for mining wheat yield-related genes and potential disease resistance proteins.The Hsp90.2-7B haplotype II works as an excellent genetic resource for future wheat breeding programs towards new varieties with high yield and enhanced disease resistance,thereby ensuring food security.